The mechanism by which human cells initiate DNA replication is unknown. Studies in the yeasts reveal that an Origin- Recognition-Complex (ORC) collaborates with CDC6/Cdc18p to initiate DNA replication. The human CDC6/Cdc18, Orc4 and Orc5 gene products were identified under the auspices of this grant. In this project these proteins will be utilized to investigate the regulation of mammalian DNA replication and to identify additional members of the human replication initiator complex. The sequence of HsCdc18 reveals biologically important sequence motifs such as a nuclear localization signal, sites of phosphorylation by cyclin-cdks and a nucleotide triphosphate binding motif. The importance of these sequences in the function of HsCdc18p and the regulation of DNA replication will be studied by a combination of biochemical, molecular biological and cell biological approaches. These experiments will indicate whether the subcellular localization of HsCdc18p or protein-protein associations are regulated by post-translational processes, and if so, whether such processes regulate S phase. HsCdc18p will be used to restore replication to an in vitro replication reaction depleted of Xenopus Cdc18p. These experiments could provide an in vitro assay for the Cdc18 protein in DNA replication and cell-cycle control. Biochemical and yeast two-hybrid approaches will be pursued to discover other cellular proteins which physically associate with HsCdc18p and HsORC subunits. These experiments will uncover additional members of the human DNA replication initiation complex and new interactions between the initiation complex and other factors involved in DNA replication and cell- cycle control. Several DNA binding assays will be used to identify human DNA sequences that interact with human initiator proteins. Such sequences could be the human replicator sequences hypothesized to exist at origins of DNA replication in human chromosomes. In the long term, these experiments will lay the foundation for understanding how DNA replication is initiated in human cells. The factors and regulatory loops involved in this process may be perturbed in pathological processes leading to human cancers. Independently, these factors and regulators will add to the repertoire of targets for developing new anti-proliferative drugs.